Our earlier studies have shown that (1) chronic treatment of rats with a benzodiazepine (BZ) decreases brain BZ receptors, indicating that these receptors may be relevant to its pharmacological effects and, (2) that there is a close functional link between BZ and Y-aminobutyric acid (GABA) receptors. The proposed research will pursue further the pharmacological and physiological significance of BZ receptors and will attempt to isolate and characterize the receptor protein. Mice will be given various doses of BZ receptor affinity ligands that will bind protein covalently (e.g. irazepine and bromoacetyl Ro7-1986/1) and the possible quantitative correlation will be sought between the number of irreversibly occupied receptors and anticonvulsant activity. Such studies will also reveal the presence of spare receptors. The synthesis rate of BZ receptors in vivo will be studied by blocking the existing receptors with i.v. or intracerebral injection of irreversible ligands to mice and measuring the initial rate of reapperance of 3H-ligand binding sites. The degradation rate of receptors that have been irreversibly tagged with (3H) or (14C)-bromoacetyl Ro7-1986/1 will be examined. The functional significance of any intracellular pool of receptors will be assessed as it relates to receptor synthesis and degradation. Drugs that affect translation or transcription will be utilized to investigate the role of protein synthesis in the regulation of receptors. The recovery and a possible rebound of BZ receptors after chronic treatment with BZs will be assessed and evaluated for possible clinical significance to rebound insomnia. BZ receptors will be solubilized with digitonin, purified by affinity chromatography, electrofocusing and other separation techniques, and their biochemical properties will be characterized.